Over the last several decades, mass spectrometry has emerged as one of the most broadly applicable analytical tools for the detection and characterization of wide classes of molecules. Mass spectrometric analysis is applicable to almost any chemical species capable of forming an ion in the gas phase, and therefore, provides perhaps the most universally applicable method of quantitative analysis. An increased amount of focus has been placed on developing mass spectrometric methods for analyzing complex mixtures of biomolecules, such as peptides, proteins, oligonucleotides, and complexes thereof. In particular, protein sequence analysis has been propelled by advances in the field of mass spectrometry (Domon et al., Review—Mass spectrometry and protein analysis. Science, 2006. 312(5771): 212-217; Ashcroft, A. E., Protein and peptide identification: the role of mass spectrometry in proteomics. Natural Product Reports, 2003. 20(2):202-215; Mann et al., Analysis of proteins and proteomes by mass spectrometry. Annual Review of Biochemistry, 2001. 70:437-473; and Coon et al., Tandem mass spectrometry for peptide and protein sequence analysis. Biotechniques, 2005. 38(4): 519, 521, 523).
Currently, analysis of proteins can be performed by a variety of electrophoresis techniques or by mass spectroscopy. Mass spectroscopy analysis is intrinsically faster and more accurate for the determination of molecular weight. In contrast to electrophoretic mobility, which is an extrinsic and highly condition-dependent property of molecules, the mass to charge ratio (m/z) utilized in mass spectroscopy is an intrinsic and condition-independent property of ions. This means that an m/z ratio determined on a mass spectrometer is intrinsically more accurate and dependable a parameter to employ for the analysis of a molecule than is the electrophoretic mobility. Second, the speed of mass spectroscopy analyses is truly phenomenal, with the potential for milliseconds per analysis. That means that, if mass spectroscopy methods for proteomics can be developed in a suitably robust and high-performance form, they have the potential to radically transform the nature of large-scale sequencing efforts.
A fundamental obstacle in mass spectroscopy has been the limited mass range accessible for the analysis of nucleic acids and proteins using Matrix-Assisted-Laser-Desorption-and-Ionization (MALDI) or Electrospray-Ionization (ESI). This limitation is manifested as a dramatic fall-off in the signal intensity with increasing mass. This phenomenon has limited the analysis of sequencing mixtures to fragment masses of less than 100 kDa, with 50 kDa being more typical. It has been shown that the fall-off in signal intensity for mixtures of large proteins is solely due to instrument-related effects, and not due to chemical issues such as ionization efficiency, or solution or gas-phase fragmentation. A thorough quantitative analysis of the instrumental issues shows that by far the most significant instrumentation issue is ion detection. A combination of the well-known decrease in secondary electron yield from microchannel or electron multiplier detectors with increasing ion mass, as well as the well-known effects of detector saturation in mixture analysis, combine to give the dramatic signal fall-off observed in the mass analysis of these complex mixtures of high molecular weight species.
Currently, among the most sensitive detectors are cryogenic calorimeters, made from superconducting junctions operating at ultra-low temperatures such as less than 100 mK (U.S. Pat. No. 5,994,694; and Twerenbold et al., Detection of Single Macromolecules Using a Cryogenic Particle Detector Coupled To A Biopolymer Mass Spectrometer. Applied Physics Letters. 68 (1996) 3503). The superconducting junction is commonly voltage-biased at its steep transition edge of the IV-characteristic, while the crystal is kept at a temperature well below the superconducting transition temperature Tc. Accelerated proteins induce showers of thermal phonons, which heat the whole crystal. This increase in temperature is monitored in the change of the IV-characteristic, since the superconducting phase transition is strongly temperature dependent. The amount of heat deposited by the phonons is directly proportional to the energy of the impinging particles. Thus these superconducting detectors possess an extremely high resolution and are able to resolve even very heavy proteins (several MDa). However, the main drawbacks of this class of detectors are the operating temperatures far below 1K and the limited number of detector pixels (maximal 16). These limitations also include slow recovery time and the fact that the individual detector elements are poorly amenable to arraying due to the complexity, i.e. number of wires to each pixel, and the need to dissipate the heat deposited into the bolometer by the molecular ions. However, even with the current progress being made, it is doubtful that such detectors will ever be operational at or close to room temperature, since they require superconducting elements. The need for cryogenic cooling increases the expense and difficulty of an experiment and is not likely to be readily adopted.
What is needed are detectors able to detect molecules that (1) are as sensitive as possible with unity detection efficiency (‘single proton resolution’), (2) do not exhibit a loss in sensitivity for large molecules, and (3) do not require cryogenic temperatures for operation. As resolution continues to be of paramount importance, it is also important that the detectors are fast, so that the process is not detector-limited in resolution.